Impact of polymorphisms of pharmacokinetics‐related genes and the inflammatory response on the metabolism of voriconazole

Abstract The effects of inflammatory responses and polymorphisms of the genes encoding cytochrome P450 (CYP) (CYP2C19 and CYP3A5), flavin‐containing monooxygenase 3 (FMO3), pregnane X receptor (NR1I2), constitutive androstane receptor (NR1I3), and CYP oxidoreductase (POR) on the ratio of voriconazole (VRCZ) N‐oxide to VRCZ (VNO/VRCZ) and steady‐state trough concentrations (C0h) of VRCZ were investigated. A total of 56 blood samples were collected from 36 Japanese patients. Results of multiple linear regression analyses demonstrated that the presence of the extensive metabolizer CYP2C19 genotype, the dose per administration, and the presence of the NR1I2 rs3814057 C/C genotype were independent factors influencing the VNO/VRCZ ratio in patients with CRP levels of less than 40 mg/L (standardized regression coefficients (SRC) = 0.448, −0.301, and 0.390, respectively; all p < .05). With regard to the concentration of VRCZ itself, in addition to the above factors, the presence of the NR1I2 rs7643645 G/G and rs3814055 T/T genotypes were found to be independent factors influencing the VRCZ C0h in these patients (SRC = −0.430, 0.424, −0.326, 0.406 and −0.455, respectively; all p < .05). On the contrary, in patients with CRP levels of at least 40 mg/L, no independent factors were found to affect VNO/VRCZ and VRCZ C0h. Inflammatory responses, and CYP2C19 and NR1I2 polymorphisms may be useful information for the individualization of VRCZ dosages.


| Patients and sample collection
Japanese patients receiving VRCZ (Vfend ® , Pfizer Japan Inc.) therapy for prophylaxis or treatment of fungal infection and who were subjected to TDM testing at Hirosaki University Hospital from January 2018 to July 2021 were enrolled in this study. TDM data of patients taking VRCZ were collected retrospectively. Patients were excluded if they (a) were aged less than 18 years; (b) were receiving dialysis; (c) did not undergo genotyping; (d) were taking drugs known to affect VRCZ pharmacokinetics. 29 Although some patients were taking proton pump inhibitors (PPIs), they were included in this analysis because of the weak effect of PPIs on VRCZ pharmacokinetics. 30 Multiple blood levels of VRCZ from the same patient were utilized in the analyses when the following patients' status changed during treatment: (a) inflammatory status (CRP ≥ 40 mg/L or < 40 mg/L); (b) route of administration of VRCZ (intravenous or oral); (c) dose of VRCZ per time. Each patient received 100 to 300 mg of VRCZ twice daily at 10:00 and 22:00 for at least 3 days. VRCZ was administered between meals to patients who were eligible for oral administration.
Initial oral or intravenous doses were determined according to the physician's clinical judgment. The target C 0h of VRCZ was 1 to 5 µg/ mL. 5 Demographic data, including age, sex, body weight, and liver and renal function tests, were collected. For age and body weight, the values at the initiation of VRCZ therapy were considered. For laboratory values, such as CRP, the values on the day TDM of VRCZ was carried out were considered.
Blood samples within 50 days after initiation of VRCZ therapy were collected in disodium EDTA tubes. These samples were collected just prior to the subsequent dosage when the plasma concentration would be at a steady state. After dose adjustment, a 3-day period was considered necessary to obtain a VRCZ C 0h steady state. 31 The VNO/VRCZ was calculated using C 0h values of the two compounds.

| Methods of analysis of plasma concentrations
Blood samples were centrifuged at 3500 rpm for 10 min at 4°C, and separated plasma was stored at −30°C until analysis. Plasma concentrations of VRCZ and VNO were measured by ultraperformance liquid chromatography (UPLC) tandem mass spectrometry using an ACQUITY UPLC System (Waters, MA, USA).
Plasma (100 μL) was mixed with 150 μL of acetonitrile and 10 μL of 10 μg/mL VRCZ-d3 as an internal standard. The mixture was vortexed for 30 s and centrifuged at 13500 rpm for 5 min at room temperature. A fraction of the supernatant (100 μL) was diluted with 200 μL MilliQ water (total volume: 300 μL). The sample was transferred to an autosampler vial, and 2 μL was injected into an ACQUITY UPLC HSS C18 column (1.8 µm, 2.1 mm × 100 mm) at for VNO, and 30V and 20 eV for the internal standard. The calibration curve was linear in the range of 0.25 to 10 μg/mL. The calibration curve showed good linearity, with R 2 > 0.99. The intra-and inter-day accuracy values, expressed as percent CV, were all within ± 15%, and precision values (as percent CV) were all less than 15% in each calibration curve.

| Genotyping
DNA was extracted from peripheral blood samples with a QIAamp Blood Kit (Qiagen, Hilden, Germany) and was stored at −30°C until analysis. The following genotypes were determined by real-time

| Statistical analysis
The Shapiro-Wilk test was used to assess distribution. Descriptive statistics of continuous variables are presented as mean ± standard deviation (SD), minimum and maximum, or median (interquartile range). Allele frequencies of polymorphisms were evaluated according to the Hardy-Weinberg equilibrium using χ 2 tests. The Kruskal-Wallis test or Mann-Whitney U test was used to determine

| RE SULTS
The demographic and clinical information of the patients at the time of the TDM are listed in Table 1. A total of 56 blood samples from 36 patients, ranging from 1 to 3 samples per patient, were obtained.
Blood samples for TDM of VRCZ were collected between 4 and 50 days after the start of administration. According to the exclusion criteria, patients taking erythromycin (n = 2) or clarithromycin (n = 2) were excluded from the study because these drugs have been demonstrated to affect VRCZ pharmacokinetics. 29 None of the patients in this study had severe liver dysfunction, as defined by a Child-Pugh score of B or C. Thirty patients were taking PPIs; 24 of these patients were taking lansoprazole. No normality was found in the distributions of the VNO/VRCZ ratio and VRCZ C 0h (Figure 1). VRCZ C 0h values outside the therapeutic range (C 0h < 1.0 µg/mL, n = 9, and C 0h > 5.0 µg/mL, n = 12) accounted for 37.5% of the total. As shown in Figure Table 2).
Analysis of variance showed that CYP2C19 EM and CRP levels less than 40 mg/L were independent factors influencing the VNO/ VRCZ ratio (both p < .01) and that there was an interaction between them (p < .05). The median ratios of the VNO/VRCZ in patients with CRP levels less than 40 mg/L were different between CYP2C19 EM

| DISCUSS ION
The results presented above suggest that the effects of CYP2C19   CYP2C19 and CYP3A4 genotypes and inflammation were shown to significantly influence VRCZ C 0h . 23 However, while these studies suggested quantitative connections among inflammation, the CYP enzymes, and VRCZ pharmacokinetics, VNO concentrations were not measured, so the impact of inflammation on the metabolic pathway was not clarified. In order to further investigate this connection, we investigated the effect of the inflammatory response on VNO/VRCZ and C 0h /D. To this end, we used a CRP cutoff value (40 mg/L) that has been established in a previous report 28  Furthermore, in addition to these factors, the NR1I2 rs7643645 and rs3814055 polymorphisms were independent factors influencing the VRCZ C 0h in these patients. PXR influences the activities of a variety of metabolic enzymes and transporters, 35,36 and it is thus not surprising that SNPs in the NR1I2 gene would affect the activity of CYP2C19 and/or CYP3A4. Notably, whereas CYP2C19 is responsible for the main pathway leading to N-oxidation, CYP3A4 appears to play a more important role in the hydroxylation pathway. 10,37 Therefore, it is possible that the effect of NR1I2 polymorphisms on the VNO/VRCZ ratio and VRCZ C 0h also reflects an effect of CYP3A4 on VRCZ metabolism.
Our results were in contrast to the results of Zeng et al., who reported that the NR1I2 rs3814057 C/C genotype increased VRCZ C 0h and the rs7643645 G/G genotype decreased VRCZ C 0h in an Asian population. 17 This discrepancy may be explained by these authors' lack of consideration of the effects of CYP2C19 polymorphisms on VRCZ C 0h . 17 In other words, it is possible that these were confounding factors. On the other hand, the work of Dapía et al., who reported that the NR1I2 rs3814055 T/T genotype decreased VRCZ AUC 0-∞ in a Spanish population, 16 was consistent with our results.
However, our work extends these results by also demonstrating the effect of the inflammatory response on VRCZ C 0h . 16  other POR variants may also affect CYP-mediated drug metabolism activities. 43 Therefore, the relationships between the pharmacokinetics of VRCZ and the FMO3 and POR polymorphisms need to be examined further.
This study had a few limitations. First, the number of patients included in our study population was small, so we may not have been able to definitively prove the effect of the NR1I2 polymorphism on VRCZ pharmacokinetic characteristics in multivariate linear regression analyses stratified by CRP. That is, it is possible that there are confounding factors. Therefore, in follow-up studies, it will be necessary to determine how the NR1I2 rs3814057, rs7643645, and rs3814055 polymorphisms affect the VRCZ C 0h in patients with CRP levels of less than 40 mg/L. Considering the non-linear pharmacokinetics of VRCZ, we predict that C 0h will be found to increase with increasing doses in patients with CRP levels of at least 40 mg/L. Second, because we did not measure the blood concentration of 4-hydoxyvoriconazole in this study, the effect of the inflammatory response on CYP3A4 activity in the metabolism of VRCZ could not be evaluated. Notably, the inflammatory response has been shown to influence the metabolic activity of both CYP3A4 and CYP2C19 42,44,45 ; in fact, Simon et al. used physiologically based pharmacokinetic models to demonstrate that CYP3A4 activity is reduced more than CYP2C19 activity when CRP levels are high. 34 Therefore, further studies exploring the effects of the inflammatory response on the hydroxylation pathway of VRCZ are needed. Third, the impacts of the CYP2C19 polymorphism on the side effects and efficacy of VRCZ therapy were not analyzed. The need for clinical trials of VRCZ therapy to determine the usefulness of CYP2C19 genotyping has long been discussed, but clinical implications of CYP2C19 polymorphism remain unresolved. 39 However, our study revealed that the impact of CYP2C19 polymorphism on VRCZ pharmacokinetic characteristics differed in the presence and absence of an inflammatory response. This result may explain the differences in degrees of the effect of CYP2C19 polymorphisms on VRCZ C 0h noted in previous reports. 46